Retrovirus derived VLPs (rVLPs) are excellent vaccine candidates, not only for retroviral diseases such as AIDS but also for a wide range of other viral diseases of clinical interest such as hepatitis C, yellow fever, West Nile fever, dengue fever, and influenza. Indeed, the retroviral core protein Gag can be modified to incorporate epitopes of interest and/or be pseudotyped with glycoproteins from a large host of families of enveloped viruses;E1E2 of hepatitis C virus, prM and E of yellow fever virus, prM and E of West Nile virus, E of Dengue virus and HA of influenza virus can all be efficiently pseudotyped onto rVLPs. Noteworthy, the pseudotyped glycoproteins retain their wild type conformation. We have engineered expression cassettes that produce rVLPs pseudotyped with one or both of the HCV envelope proteins E1 and E2 (HCV-E1-rVLP or HCV-E1E2-rVLP). We have shown that mice primed with a recombinant viral vector expressing the HCV envelope proteins E1 and E2 will mount unprecedented neutralizing humoral immune responses upon boosting with pseudotyped rVLPs, compared with a second immunization with the recombinant viral vector. However, while these pseudotyped rVLPs make highly promising candidates for a vaccine against Hepatitis C, they are cumbersome to produce and thus their production calls for a new efficient and safe system. We have previously compared HCV-rVLPs produced via baculovirus vectors in an insect cell line or via DNA vectors in a human cell line. The baculovirus vector system gives rise to considerably higher HCV-rVLP titers than the DNA vector system. However, HCV-rVLPS produced from the DNA vector system induce far better humoral immune responses, likely due to a correct glycosylation pattern provided by the human cell line. We believe that a lentivirus vector system could alleviate the production bottleneck. Indeed, lentiviral vectors can be stably incorporated at high copy numbers per cell, and they are readily expressed in human cell lines. Therefore, the aim of this grant application is the construction of a lentiviral vector expressing HCVrVLPs, and validation of its use for high titer production of HCV-rVLPs from human cells stably transduced with the lentiviral vector. Such a system would pave the way for continued development of the Hepatitis C vaccine candidate presented here, as well as for any vaccine candidate consisting of rVLPs. PUBLIC HEALTH RELEVANCE: The Heptaitis C virus is the most common blood-borne virus and HCV infection represents a major public health concern;approximately 3% of the world's population (200 million people) is chronically infected. Causing fibrosis and cirrhosis of the liver and eventually hepatocellular carcinoma, it is the leading cause of liver transplantation in the U.S. No vaccine is available and the current standard of care, a combination of Pegylated-Interferon alpha and the antiviral drug Ribavirin, is effective in only about 50% of patients completing therapy, is lengthy, and is often poorly tolerated. New anti-viral drugs are in the early clinical stage of development, but the available pre-clinical and clinical data indicate that drugresistant variants are likely to emerge during treatment. Thus, the patient population is still underserved and treatment needs remain unmet. Therefore, there is a strong need for new alternative therapies that act through different mechanisms, such as prophylactic and therapeutic vaccines. Lentigen has developed a novel LENTIMAXTM Lentiviral vector gene delivery system that can deliver payload with 100% efficiency in mammalian cells, resulting in very high levels of protein production. Lentiviral vectors (LVs) have recently been shown to achieve HIGH and STABLE gene delivery in a variety of cell lines used for the manufacture of proteins and vaccines. Therefore they are the ideal system for the rapid and efficient delivery of genes encoding proteins into cells for high yielding production of target proteins. Lentigen has exclusive intellectual property in the production of proteins using Lentiviral vector technology. Lentigen's LENTIMAX(tm) system is inherently flexible in that any cell line can be efficiently and stably engineered with any gene(s) of interest into any mammalian cell type. The technology can be used in multiple formats, and in particular related to this RFA it can be used for rapid production of virus-like particles from human cells.